Culture-Free Next Generation Sequencing for Rapid Detection of Drug-Resistant Tuberculosis
Funding Source: NIH 5R21AI135756-02
We propose to leverage an existing NIH research collaboration between UCSD, TGen, UARK, and the Moldova National Tuberculosis Program to gather performance data on a Next Generation Sequencing (NGS) assay, which detects clinically relevant drug resistance tuberculosis (TB) directly from patient sputa.
TB has continued to be a global epidemic despite 60 years of advances in the fields of drug therapy, microbiology, diagnostics, genetics, and epidemiology. In 2016, the World Health Organization (WHO) estimated there were 10.4 million new TB cases, of which 580,000 were multi-drug resistant (MDR)-TB.1 Patients with any form of drug-resistant (DR) TB have a much greater risk of dying than those with drug susceptible TB;2 with resistance to 2nd-line anti-TB drugs increasing this risk more than fourfold.3-5
The cornerstone of diagnosing TB has been through laboratory confirmation which combines microscopic examination of sputum with culturing (growing) samples,6 and the detection of DR-TB through culture-based drug susceptibility testing (DST) remains the most widely used WHO reference standard.7 However, despite decades of efforts to establish culture-based DST around the world, less than 60% of all TB cases are confirmed using laboratory methods and many countries still have no capacity to perform 1st or 2nd line anti-TB DSTs.1 There are complex reasons for this laboratory capability gap,8, 9 but one reason is there is a shortage of cost-effective methods for rapidly detecting 1st or 2nd line drug resistance that are not dependent on the expensive and slow culture-based methods.8, 9 Our long term goal is to develop a high-throughput sequencing-based DST which requires no complex biosafety facilities, significantly accelerates drug resistance results, and moves away from reliance on culture.
Our group has developed a promising new approach that uses targeted NGS on clinical specimens, with no intermediate culture step, to detect mutations in Mycobacterium tuberculosis (Mtb) that confer resistance to existing 1st and 2nd-line anti-TB drugs used in: (a) standard TB therapy,10 (b) established MDR-TB therapy,11 (c) the newly endorsed MDR-TB short-course therapy,6 and (d) novel regimens currently undergoing clinical trials.12 Our simplified assay (NextGen-RDST) is run on the widely available Illumina MiSeq instrument and is suitable for implementation in reference laboratories in low and middle income countries13 where short course MDR-TB treatment is currently being rolled out. Our preliminary studies, based on an initial set of remnant DNA from MDR-TB sputa, have demonstrated excellent performance for detecting MDR-TB (sensitivity of 97.6%, specificity of 98.9%) as well as XDR-TB (sensitivity of 90.0%, specificity of 97.8%) within 48 hours.14 Further evaluation of our assay, using prospective, sequentially collected clinical samples is needed prior to any large-scale clinical trial. The objective of this proposal is to leverage well-characterized clinical sputum samples collected from an existing project examining patients at risk for MDR-TB in Moldova (R01AI111435) to assess the performance of our culture-free, NextGen-RDST assay by comparing our results to genotypic and phenotypic reference standards. We propose to accomplish this with the following specific aims:
Specific Aim 1: Determine the performance of our culture-free NextGen-RDST relative to the reference phenotypic DST using samples collected from 500 patients at risk for MDR-TB. Hypothesis: Based on preliminary studies, we hypothesize that targeted NGS direct from sputum will accurately detect 1st and 2nd line drug resistance with ≥90% sensitivity and ≥98% specificity to phenotypic DST.
Specific Aim 2: Determine the performance of our culture-free NextGen-RDST relative to the reference genomic sequencing of samples collected from 500 patients at risk for MDR-TB. Hypothesis: Mutations identified with targeted NGS direct from sputum will have ≥95% sensitivity and ≥95% specificity when compared to mutations identified by whole genome sequencing (a genotypic reference standard) performed on cultures obtained from the same clinical samples.
Specific Aim 3: Analyze factors contributing to discordance between NextGen-RDST and phenotypic and genotypic reference standards. Hypothesis: Discordance between the direct NextGen-RDST assay and indirect reference methods used to validate their performance is driven primarily by absence of expected resistance alleles, differences in assay sensitivity to micro-populations of resistant organisms and/or changes in heteroresistance sub-population proportions introduced during culture (a required step for phenotypic and whole genome sequencing reference methods).
Our central hypothesis is that NextGen-RDST is an accurate and reliable, high-throughput assay for detecting DR-TB. For this R21, we propose to gather sufficient evidence of the assay’s performance to support a subsequent multisite clinical trial of NextGen-RDST. The study leverages an existing NIH research collaboration between UCSD, TGen, UARK, and Moldova NTP and will contribute novel and generalizable information regarding the primary causes of discordance between direct molecular DST and the indirect standard reference methods we use to validate them.
References
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